USPTO Patents Application 10687850

PCT

WORLD INTELLECTUAL PROPERTY ORGANIZATION
International Bureau

mm

INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)

(51) International Patent Classification 5 :

G01N 33/543, C12M 1/40, C12Q 1/00

Al

(11) International Publication Number: WO 94/28414

(43) International Publication Date: 8 December 1994 (08.1254)

(21) International Application Number: PCT/EP94/D1714

(22) International Filing Date: 26 May 1994 (26.05.94)

(30) Priority Data:

9311206.8
9325898.6

29 May 1993 (29.05.93) GB
17 December 1993 (17.12.93) GB

(71) Applicant (for all designated States except US): CAMBRIDGE

LIFE SCIENCES PLC [GB/GBJ; Cambridgeshire Business
Park, Angel Drove, Ely, Cambridgeshire CB7 4DT (GB).

(72) Inventors; and

(75) Inventors/AppBcants (for US only): ATHEY, Dale [GB/GB];
53 Ascot Walk, Kingston Park, Newcastle-upon-Tyne NE3
2UG (GB). McNEIL, Cahnn, J. [GB/GB]; 20 Word Road,
Jesmond, Newcastle-upon-Tyne NE2 3NX (GB). ARM-
STRONG, Ronald, D. [GB/GBJ; 73 Grosvenor Avenue,
Newcastle-upon-Tyne NE2 2NQ (GB). MULLEN, William,
Henry [GB/USJ; 833 Deep Wood Court, Grayslake, DL
60030 (US).

(74) Agent: RUPP, Herbert; Byk Gulden Lomberg Chemische
Fabrik GmbH, P.O. Box 100310, D-78403 Konstanz (DE).

(81) Designated States: JfP, US, European patent (AT, BE, CH, DE,
DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE).

Published

With international search report

Before the expiration of the time limit for amending the
claims and to be republished in the event of the receipt of

(54) Title: SENSORS BASED ON POLYMER TRANSFORMATION
(57) Abstract

A sensor format is described based on the impedance analysis of polymer coatings of electrodes. The detectable signal is produced
by the effect of a reactive or catalytic species at or very near to the polymer coated electrode. Said reactive or catalytic species directly
or indirectly effects a reaction with the said polymer layer wherein the polymer layer becomes porous causing a measurable change in
electrical properties at the electrode surface.

WO 94/28414

PCT/EP94/01714

Sensors based on Polymer Transformation
Background of the Invention

The concept of sensors based on an electrochemical transducer sensitised with a biological
moiety, such as an enzyme, is both simple and elegant and offers the prospect of reagendess
clinical analysis with minimum sample preparation. The major advantage of this approach for
medical use is ease of operation, thus obviating the requirement for trained laboratory personnel
to carry out the measurement. This should allow deployment of sensors in decentralised
laboratories and facilitate a more rapid return of clinical information to the clinician. The net
benefit being an earlier institution of appropriate therapy [Recent Advances in Clinical Chemistry,
Vol.3, Alberti, KGiMM and Price, CP (eds), Churchill Livingston 1985]. However, in few cases '
has the concept been translated into practical working devices suitable for near patient testing.
Commercially available biosensors based on electrohemical methods are generally either
potentiometric or amperometric and for certain clinically important analytes, both of these
techniques have drawbacks for biosensor exploitation. In order to circumvent these problems and
to produce specific, sensitive techniques suitable for deployment in decentralised laboratories we
have demonstrated the feasibility of constructing sensors based on polymer membranes which are
modified during operation due to the changes in pH and lead to highly sensitive changes in
impedance at underlying electrodes.

Techniques in relation to this type of process have been disclosed for instance, in US Patent 4 352
884 and EP 0 31 1 768. US Patent 4 352 884 describes a pH electrode having an acrylate
copolymer coating for the inunobilisarion of bioactive materials which was used for the
measurement of urea.

This method is only for the immobilisation of bioactive materials such as antigens, antibodies and
enzymes and does not play a part in the measurement process. The use of polymer coatings on
pH electrodes has a disadvantage of a small dynamic range and poor sensitivity.

EP 0 3 1 1 768 describes the modification of a semi-conductive polymer coated on the electrode
which becomes more conductive as a result of a homogeneous immunoassay using enzyme
conjugates. The method is based on the measurement of resistance which applies a large voltage
(500 mV) to the system which may perturb it. The method appears only to cause changes up to* 1
order of magnitude and is therefore not as sensitive as the method disclosed in the present
invention.

In the present invention, a novel sensor format is described based on the impedance analysis of
polymer coatings on electrodes, which require simple fabrication and measurement techniques.

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PCT/EP94/01714

The impedance of an electrode is sensitive to a number of factors. Changes in electrode
impedance are often caused by changes in double layer capacity. The double layer capacity (Cdl)
arises from the separation at the surface of an electrode between the electronic charges in the
metal and the (mobile) ionic charges in the solution in contact with the metal. For a metal (e.g.
gold) in contact with a solution of aqueous potassium chloride, this separation is due to the
presence of a layer of water molecules on the metal surface. This double layer capacity may be
calculated from the standard formula for a parallel plate condenser,

Cdl = e o e rA/d (i)

where

e r is the effective dielectric constant of the water layer,

A is the electrode area and

d is the diameter of a water molecule.

Tf instead of a layer of water molecules, there is an insulating polymer layer between the metal
and the aqueous solution, equation (1) still holds, but d represents the thickness of the polymer
layer.

It has now been found that the production of minute imperfections in such a polymer layer give
rise to a dramatic increase in capacity which can easily be measured.

The impedance of an electrode is determined by applying a sinusoidal potential of small peak to
peak amplitude (typically <10 mV) to the electrode and measuring the resultant sinusoidal
current The range of frequencies which are employed lie between 1()5 Hz and 10-3 Hz. There is
generally a phase difference (q) between the potential and current so that the ratio of potential to
cunent is essentially a vector quantity (Z) which has magnitude (El) and direction (q).
Impedance measurements are often represented in the complex impedance plane where the two
components of the impedance vector (Z* and Z") are plotted against each other with frequency as
a parameter.

For the electrode impedance to be well defined, it must be measured under steady state
conditions. If the electrode condition is changing, the impedance will only be well defined at
frequencies above a frequency which is dependent on the rate of change. In principle, an
impedance measurement can be made when a steady state current is flowing due to the oxidation
or reduction of a species in the solution.

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PCT/EP94/01714

Summary of Invention

The impedance of an electrode can be changed in many ways. For example, the adsorption of a
protein on an electrode will cause the'electrode impedance to change. However, in order to be
useful as a sensor the change of impedance must be highly specific to the substance being sensed
and give high sensitivity.

The current invention relies on the occurrence of ;tn enzynric reaction creating changes in the
impedance of the electrode as a result of the partial or complete removal of an insulating polymer
film from its surface. Other methods of measuring the results of the polymer transformation can
be envisaged, such as the measurement of increased current at a polarised electrode. The enzyme
or catalyst produces a product which can react with the polymer, or can directly hydrolyse the
polymer membrane.

The said enzymes or catalysts may be bound within or direcdy to the polymer, be present in the
bulk solution or bound to the polymer via an antibody-antigen interaction with an enzyme-labelled
conjugate or bound to a porous membrane in close proximity to the polymer coated electrode.
The examples should not be construed to be the only possible formats of this system.

A further possible application is to transform the polymer coating by an enzymic reaction which
causes a change in pH which then solubilises the polymer allowing the passage of electrolyte to
the underlying electrode resulting in a large change in impedance.

One of many appropriate combinations of an enzyme with a polymer coating is the combination
of urease with materials such as those used in enteric coatings for tablets. These coatings work
by being insohible at the low pH of the stomach, but are soluble at the pH in the intestine (pH 6
and above). Examples of such coating materials are cellulose acetate hydrogen phthalate, methyl
vinyl ether-maleic anhydride copolymer esters, anionic polymerisates of methacrylic acid and
esters of methacrylic acid (Eudragit® of Rdhm-Pharma, Darmstadt, Germany).
In one particular reaction, urease catalyses the breakdown of urea to ammonia and carbon dioxide
according to the following scheme:

H 2 0 + urea -> 2NH 3 + CO2

NH 3 + H 2 0 -> NH4+ + OH-

The resulting increase of the pH value leads to a solubilisation of said materials.

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PCT/EP94/01714

Another application is to apply a P H sensitive polymer whose permeability is reversibly regulated
by pH changes in the outer medium. The polymer, poly 4-vinylpyridine is coated on the electrode
surface and when the pH becomes acidic, the permeation to ions is increased which is measured
by an increase in capacitance at the electrode. Many enzyme reactions may cause the pH to
become acidic. In one particular reaction, glucose oxidase catalyses the breakdown of glucose to
gluconolactone and peroxide according to the following scheme:

Glucose + O2 -» gluconolactone + H2O2
The permeability of the polymer is reversible and can be re-used many times.

If an antibody is immobilised onto said pH-sensitive polymers, it may capture an urease-labelled
conjugate in an immunoassay. The bound urease conjugate then produces a local pH change that
leads to the sdlubilisation of the polymer at the point of conjugate capture. Most unexpectedly
we have found that local solubilisation can occur in solutions where, because of the buffering
capacity of the bulk solution, a significant pH change in the solution does not take place.

It is also possible toenhance this method by polarising the working electrode at a potential where
reacnve species can be produced when the electrode comes into contact with the electrolyte If
an electrode is polarised at -600 mV vs Ag/AgCl no reaction will occur until some small amount
of electrolyte comes into contact with the electrode, as the polymer layer is just starting to break
down. Oxygen reduction at the electrode surface then produces OH" which increases the local
pH further and accelerates the breakdown of the polymer. This results in considerable
amplification of the original signal

In another instance it is possible to use an ^Oz-producing enzyme such as glucose oxidase in
conjunction with the Fenton reaction. This reaction is commonly used in synthetic organic
chemistry to hydroxylate aromatics; and works by producing the extremely reactive radical OH.-
e.g.

H2O2 + Fe2+ -» Fe3+ + OH" + HO.

The hydroxyl radical is so reacnve that it survives only until it encounters an organic species. In
this case a polymer coating is chosen which contains structure elements reacting with the HO
radicals. The introduction of hydroxyl groups enhances the solubility of the coating giving rise to
a significant increase of the double layer capacity.

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PCT/EP94/01714

Examples

The following examples are provided to further illustrate the present invention. It is to be
understood that the examples are for purpose of illustration only and are not intended as a
definition of the limits of the invention.

General mechanism

Planar carbon electrodes (1 mm diameter) formed by screen printing were coated with cellulose
acetate phthalate (CAP) (Kodak) as follows:

A 70 mg sample of CAP was mixed with 30 mg of diethyl phthalate plasticiser and added to 400
mg of acetone to form a viscous solution. A 5 pi aliquot of this solution was then placed onto the
working electrode and allowed to dry in air at room temperature.

The capacitance of the coated electrodes was measured in 150 mmol l" 1 sodium chloride, pH4,
using a frequency response analyser (Schlumberger) and compared with that of bare carbon
electrodes. Coated electrodes were then exposed to 150 mmol H sodium chloride, pH 6.5, for
15 minutes and the capacitance re-measured.

Electrode Condition,
Bare electrode capacitance

Caoacitance (pFcm' 2 )
24.6

Coated electrode capacitance, pH 4

0.002

Coated electrode after 15 minutes at pH 6.5

0.331

Exposure of the coated electrode to a solution at pH 6.5 resulted in a 160 fold increase in the
capacitance of the electrode.

Gold rod electrodes (4 mm diameter) were coated with cellulose acetate phthalate (CAP)
(Kodak) as follows:

The electrode was dip coated once using a 1:8 w/w CAP / 30 % diethyl phthalate plasticiser to
acetone mixture and allowed to dry for 30 minutes at room temperature.

The capacitance of the coated electrodes was measured in 140 mmol H sodium chloride, pH4,
using a frequency response analyser (Schlumberger) in a three electrode configuration using a
silver/silver chloride reference and gold counter electrode. The capacitance of the coated

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PC17EP94/01714

electrode was measured after 5 minutes and 1 hour. The pH 4 solution was removed from the
cell and replaced with pH 6.5, 140 mmol l" 1 sodium chloride solution and the capacitance
measured at 30 minute intervals.

CaDacitanrpffiFcm" 2 )

Uncoated electrode

12.2

Coated electrode, pH 4, 5 minutes

0.002

Coated electrode, pH 4, 1 hour

0.002

Coated electrode, pH 6.5, 30 minutes

_ 1.176

Coated electrode, pH 6,5, 1 hour

1.584

Coated electrode, pH 6.5. 1.5 hours

1.944 1

Coated electrode, pH 6.5, 2 hours

2.224 •

These results are represented graphically in Figure 1.

the pK a values of enteric coatings, CAP and Eudragit S 100 polymers, have been calculated by
titration of the solid in aqueous solution against dilute base. As both polymers* pH sensitivity is a
direct result of the deprotonation of acid ester groups, the pK a value corresponds to the
breakdown pH of the polymer. The pK a of CAP is approximately 6.0, and the pK a of Eudragit
S100 approximately 7.0.

B REAKDOWN OF CELLULOSE ACETATE PHTHALATE FILM USING UREASE

A gold rod electrode (4 mm diameter) was coated with CAP as previously described and inserted
into a glass cell The buffer solution (1 ml) was EDTA (0.5 mM), sodium chloride
(140 mM), urea (16 mM) adjusted to pH 4.8 using hydrochloric acid (0.1 M).

Capacitance values were obtained at pH 4.8 after 30 and 60 minutes and then urease (20 ^1 of a 1
mg ml" 1 solution) was added and the capacitance measured at 90, 120 and 150 minutes.

Time(min)

CaDacitance (^p cm* 2 )

30

0.0004

60 (urease added)

0.0004

90

0.0133

120

5.000

150

.6.500

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PCT/EP94/01714

These results are represented graphically in Figure 2 and confirm that it is possible to breakdown
the CAP polymer film by the use of the enzyme urease causing a change in pH and resulting a a 4
orders of magnitude change in electrode capacitance.

Breakdown of Eudragit S 100 polymer using urease.

The gold rod electrodes (4 mm diameter) were dip coated twice using 20% w/w Eudragit S100
(Rohm Pharma) in acetone containing 20% w/w dibutyl phthalate plasticiser and 5% w/w Tween
80 and then placed in an atmosphere saturated with acetone for 15 minutes. The electrodes were
then removed and allowed to dry for 30 minutes at room temperature.

The glass cell was used containing 1ml of buffer solution composing of 0.2 mM EDTA, 0.15M
NaCl, pH 5.8 and urea over the range 5 - 50 mM. Initial capacitance values were obtained and
after the addition of urease (0.1 mg mH). The rate of change in impedance as a function of time
was urea concentration dependent. The ratio of the capacitance after 10 minutes to the initial
capacitance (CtfCo) is shown as a function of urea concentration in Figure 3. This clearly
demonstrated that urea can be determined by thii method and that large changes in signal are
involved.

Threshold/time breakdown of Eudragit S 100 polymer due to urea concentration.

Stainless steel discs, 10 mm diameter, were sprayed with Eudragit SI 00 polymer. The polymer
composition was l.lg Eudragit'SlOO (Rohm Pharma) dissolved in 13.7g acetone. Dibutyl
phthalate (0.25g) and Tween 80 (0.3g) were then added to the mixture. This was applied to the
surface of the stainless steel electrodes using a spray nozzle attached to a compressed air cylinder
(20 lbf/in 2 ). The optimum spray conditions proved to be 2 applications of polymer with a 30
minute drying time between coats. '

Immunodyne niembranes (Pall Biosupport) of 0.38 cm* were loaded with 5 mg ml" 1 urease in
PBS by applying 50 ftl to the surface of the membrane for 16 hours at 4*C. The membranes were
washed with 140 mM Nad, 0.2 mM EDTA solution.

Membranes were placed onto the surface of the polymer coated electrode, a gasket placed over
the surface of the membrane/electrode and placed in the electrochemical cell. The cell was filled
with 1 ml of 140 mM NaCl, 0.2 mM EDTA, pH 5.8 solution.

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PCT/EP94/01714

Impedance measurements were made over the frequency range 20kHz to lOmHz to test the
integrity of the polymer film. Once the films had been tested, the experiment was started by
adding the appropriate amount of urea. The capacitance was followed with time at a frequency
of 20kHz, until the polymer film had disintegrated.

The results which are shown in Figures 4 and 5 clearly demonstrate the basis of a 'threshold'
measurement for urea.

Poly (4-vtnyl pyridine) (PVP) coatings on organometallic electrodes

Experiments were performed to test the use of PVP as a polymer coating which would break
down at acid pH.

Organometallic gold electrodes were^dip coated (xl) with the polymerias supplied in methanol
from Aldrich, Chem Co, UK), the electrodes were then left to dry for 16 hours.

The electrodes were then tested as follows:

Impedance measurements were made over the frequency range 20kHz to 1Hz, and capacitance
values calculated at 20kHz. Electrodes were placed in the cell with 0.1M carbonate buffer
pH9.6, and an initial capacitance measurement made. A value was then taken after 60min.
Subsequendy, the buffer was replaced by 0.2mM EDTA/ 140mM Nad buffer, pH 5.8.
Capacitance measurements were made after 5min and 30min. Finally, 0.1M carbonate buffer pH
9.6 was re-introduced into the cell and further capacitance measurements made at 5min and
30min.

Figure 6 clearly shows the pH dependency of the insulating properties of the polymer coating,
and the reversible nature of this pH effect

Biotin Immunoassay based on Polymer Breakdown

A model experiment was performed to demonstrate the immunoassay principle.

Avidin coated membranes were prepared as follows; A 5 x 5cm piece of nitrocellulose was
wetted using H 2 0 and then placed on a piece of WTjatman No.l filter paper, and saturated with
lml of 5mg ml-1 avidin solution in PBS, pH 7.4. After lh the membrane was rinsed in PBS and

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PCT7EP94/01714

10ml of 0.1% glutaraldehyde in PBS added. After 2h the membrane was rinsed in water, and
PBS.

Membrane discs were then incubated with biotin standards (200(jJ, 0 to lOOjlg mH in PBS
pH7.4) and a biotin urease (Biogenesis, UK) conjugate (200|xl, 1/50 dilution in PBS
pH 7.4) for 2h at room temperature. The membranes were then removed, washed in water
twice, and 0.2mM EDTA/140mM NaCl solution once. The membranes were then placed in the
electrochemical cell, over the top of an Eudragit S100 polymer coated stainless steel disc to
perfomi impedance analysis.

The urea concentration was adjusted to lOOmM by the addition of 100^1 1M urea to the 900^1
NaCl/EDTA solution in the cell, and mixing with a pipette. After addition of urea the impedance
was monitored for lh, capacitance measurements were made from the impedance values at
20kHz every 15min.

Biotin

Capacitance (nF)

Capacitance (nF)

6C(nF/h)

Hgml-1

time = 0

time = 1 hour

0

14.6

141.3

126

0.01

25.3

13217

107

1

13.4

52.7

39

100

26.8

23.3

0

The electrochemical assay according to this invention can be used in many different formats
known to the man skilled in the art

So it is possible to immobilise in any manner known to die man skilled in the art an antibody at
the electrode surface for which the analyte to be measured and an analyte enzyme conjugate or an
analyte analogue enzyme conjugate compete.

For a sandwich format a first antibody against the analyte to be measured is immobilised at the
electrode surface and a second antibody labelled with an enzyme is present in the solution.

For a competition format, a capture antibody is immobilised on a solid phase and then a sample is
added containing labelled and unlabelled antigen which compete for antibody sites on the solid
phase. The amount of label revealed is inversely proportional to the amount of antigen in the
sample.

WO 94/28414 ^ ^ PCT/EF94/01714

In a competition fotmat the solution contains a biotin labeUed analyte or a biotin labelled analogue
of the analyte. An enzyme conjugate with avidin is also present in the solution or may be added
after the capture reaction of the biotin labelled amdyte or analyte analogue and the analyte to be
measured with the immobilised antibody. Other binding pairs in place of avidin/biotin, e.g.
IgG:anti-IgG may be used equally.

It is also possible to use a competitive assay where an analyte or an analogue of the analyte is
conjugated with an anti-enzyme antibody. Also present in solution is the analyte to be measured
and free enzyme, where the signal generated is inversely proportional to the analyte concentration
being measured.

Either a sandwich assay format or competition format can also be used where the solid phase is
either a coated tube or microwell plate. In this format, the polymer coated electrode is dipped
into the coated tube or microwell where the enzyme conjugate reaction with substrate causes the
transformation of the polymer.

In a further format, the competition between the analyte and an enzyme conjugate of the analyte
or an analogue of the analyte with an enzyme can be performed in a wick (bibulous layer) or a
capillary channel in which an antibody against the analyte is immobilised on the surface. After
having passed the wick or capillary channel, the unbound enzyme conjugate of the analyte comes
into contact with the electrode where an anti-enzyme antibody is immobilised. The signal
generated is proportional to the concentration of analyte present

Description of Figures

The invention is illustrated by the figures 1 to 6:

Figure 1 is showing capacitance results from (a) uncoated gold electrode (b) CAP coated gold
electrode, pH4.0 and (c) CAP coated gold electrode after immersion in pH6.5 buffer.

Figure 2 is showing capacitance of CAP coated gold electrode as a function of time and the
effect of urease (20 |iL of lmg/mL) addition.

Figure 3 is showing capacitance change for different analyte (urea)

concentranon.

Figures 4 and 5 are showing the breakdown of polymer coated electrodes due to analyte (urea)
concentration with respect to time.

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PCT/EP94/01714

Figure 6 is showing the effect of solution pH on the capacitance of a poly(4-vinyl pyridine)
coated electrode with time. The electrode at t = 0 mins is in carbonate buffer pH9.6. At (a) the
electrode is in NaCI/EDTA pH5.8 (t = 60 mins) before returning (b) to carbonate buffer pH9.6
(t = 95 mins).

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PCT/EP94/01714

Claims

1 . A method for determining the presence of an analyte in a sample suspected of containing
said analyte, said analyte reacting with an enzyme or catalyst wherein the amount of
detectable signal is a function of the amount of analyte in the assay medium, said
detectable signal being detected at an electrode, where

a) the electrode is covered with a thin layer of an electrically insulating polymer that
separates the electrode from an electrolyte;

b) the enzyme or catalytic reaction with the said analyte direcdy or indirectly effects a
reaction with the said electrically insulating polymer layer wherein the polymer
layer becomes porous or more porous to the electrolyte, causing a measurable
change in electrical properties at the electrode surface.

2. A method according to claim 1 where the electrically insulating material is a pH-
sensitive polymer and where the reactive or catalytic species causes a change in pH
sufficient to increase the permeability of the electrode covering layer.

3. A method according to claim 2 in which the reactive or catalytic species is an enzyme
that reacts to cause a change in plL

1 A method according to claim 2 in which the reactive catalytic species is urease or
glucose oxidase.

5. An immunoassay method in which a enzyme-antibody or enzyme-antigen conjugate
becomes bound at or very near a layer of polymer via a specific immunochemical
reaction where the layer of polymer covers an electrode and where die enzyme
activity of the conjugate leads to an increased permeability of the polymer layer to
electrolyte, measured by a change in capacitance or current passed by the underlying
electrode.

>. A methdd according to claim 1 in which the electrode is polarised at a potential at
which a redox reaction can occur with a constituent of the electrolyte such that
breakdown of the polymer layer becomes accelerated once the electrode first
becomes exposed to the electrolyte.

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PCT/EP94/01714

7. A method according to claim 1 where the activity of the catalytic species results in
the formation of a hydroxyl radical, which is able to react with the polymer layer
resulting in an increase in the permeability to electrolyte of said layer.

8. A method according to claim 7 where the catalytic species is a hydrogen peroxide-
producing oxidase and the electrolyte contains species able to promote the Fenton
reaction.

9. A method according to claim 1 in which the catalytic species is a polymer-degrading
enzyme which acts directly on the polymer layer to increase the permeability of the
layer.

10. A method accoiding to claim 9 in which the catalytic species is amylase or
amyloglucosidase and the dielectric comprises or contains a polysaccharide.

11. A method according to claim 9 in which the catalytic species is an endonuclease and
the polymer comprises or contains a nucleic acid.

12. A method according to claim 9 in which the catalytic species is a lipase and the
polymer comprises or contains lipid.

13. A method according to claim 1 where the enzyme or catalyst is within the polymer
layer.

14. A method according to claim 1 where the enzyme or catalyst is bound to the polymer
layer.

15. A method according to claim 1 where the enzyme or catalyst is bound to another
support in close proximity to the polymer layer such as nitrocellulose membrane or
other membrane support material

16. A method according to claim 1 where the sample is aqueous, whole blood, serum,
plasma, urine or saliva.

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2/6

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PCT/EP94/01714

Fig. 3

100000

Urea concentration (mM)

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PO7EP94/01714

Fig. 4

icr 5 -3

O 10 20 30 40 50 60 70 80 90 100 110 120 130

Time (min)

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PCT/EP94/01714

Fig. 5

Urea Concentration (mM)

Time (min)

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PCT/EP94/01714

Fig- 6

o

to
a.

O

io- 7 ^

10

-8

10

-9

t = 0 rains (carbonate buffer pH9.6)

(a) t = 60 mins (NaCl/EDTA pH5.8)

(b) t = 95 mins (carbonate buffer pH9.6)

-, — | — , — r
0 25 50

T

T

T

75 100 125 150
Time (min)

175 200

INTERNATIONAL SEARCH REPORT

IntcmatK Application No

PCT/EP 94/01714

a . cxassii k;a tion of suhjkct maitkr
IPC 5 G01N33/543 C12M1/40

C12Q1/00

According in International Patent gasification (11*0 or to both national classification and IPC

n. MKI.PS siiARcimp

Minimum documenlaUon searched (classificaUon system followed by classification symbols)

IPC 5 G01N C12Q

Documentation searched other than minimum documentation to the extent that such documents arc included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Citation of document, with indication, where appropriate, of the relevant passages

Relevant to claim No.

ANALYTICAL CHEMISTRY,

vol.64, no. 21, 1 November 1992

pages 2645 - 2646

O.T.HOA ET AL. 'Biosensor Based on

Conducting Polymers'

see the whole document

EP.A.O 467 219 (MILES INC.) 22 January
1992

see page 1 - page 11; claims

W0.A.93 06237 (ALLAGE ASSOCIATES, INC.) 1
April 1993

see page 1 - page 11; claims; example 4

W0.A.90 10655 (ALLAGE ASSOCIATES, INC.) 20
September 1990

see page 13 - page 20; claims

-/~

1-3,7,8,
13,16

1-4,13,
16

1-5,7,8,
13,14,16

1-4,7,8,
14,16

El

Further documents arc listed in the continuation of box C

0

Patent family members arc listed in annex.

* Special categories of cited c

'A* document defining the general state of the art which is not
considered to be of particular relevance

*R* earlier document but published on or after the international
filing date

"1/ document which may throw doubts on prion ly daimfs) or
which is cited to establish the publication dale of another
citation or other special reason (as specified)

"O* document referring to an oral disclosure, use, exhibition or

P" document published prior to the international firing date but
later than the priority date claimed

T" later document published after the international filing date
or priority date and not in conflict with the application but
a ted to understand the principle or theory underlying the
invention

'X* document of particular relevance; the claimed invention
cannot be considered novel or cannot be considered to
involve an inventive step when the document is taken alone

"Y* document of particular relevance; the d aimed invention
cannot be considered to involve an inventive step when the
document is combined with one or more other such docu-
ments, such combination being obvious to a person skilled
in the art.

document member of the same patent family

Date of the actual completion of the international search

26 September 1994

Date of mailing of the international search report

1 1. 10. 94

Name and mailing address of the ISA

Kuropcan Patent Office, P.B. 5818 Patentlaan 2
Nl, - 2280 HV Rijswijk
Tel. ( » 31.70) 340-2040, Tx. 31 651 cpo nl.
Far < * 31-70) 340-3016

Authorized officer

Hitchen, C

Fbtm PCT/lSA/210 (nxsod sfaed) (Jury IW2>

page 1 of 2

INTERNATIONAL SEARCH REPORT

Internal Application No

PCT/EP 94/01714

CfConunuation) DOCUMENTS CONSIDERED TO BE RELEVANT

Category * Q taa on of document, with indication, where appropriate, of Ihc relevant passages

Relevant to claim No.

EP.A.O 314 009 (MILES INC.) 3 May 1989

See page 11; page 27-page 28; Page 34-page
35; claims.

EP,A,0 311 768 (0HMICR0N CORPORATION) 19
April 1987

cited in the application
see the whole document

W0.A.86 04926 (GENETICS INTERNATIONAL
INC.) 28 August 1986
see the whole document

W0,A,89 01159 (COMMONWEALTH SCIENTIFIC AND
INDUSTRIAL RESEARCH ORGANISATION) 9
February 1989

1-4,7,8,
14,16

1,5,14,
16

1,4,
9-12,16

1,5

Form PCT7ISA/210 (oonunotfon of second *hc*t) (July 1992)

page 2 of 2

INTERNATIONAL SEARCH REPORT

Imu nation on patent family members

Palcnt document

Publication

Patent family

Publication

cited in search report

date

membcr(s)

date

EP-A-0467219

22-01-92

AU-B-

635432

18-03-93

All A

AU-A-

8029991

23-01-92

CA-A-

2043807

20-01-92

JP-A-

6022793

01-02-94

US-A-

5202261

13-04-93

US-A-

5250439

WO-A-9306237

Ul U*t 170

US-A-

5312762

cv Uj jU

US-A-

5312762

1 7— nc_ OA

EP-A-03 14009

03-05-89

US-A-

4886625

12-12-89

AU-A-

2433088

13-07-89

AU-B-

626737

06-08-92

AU-A-

6650790

14-03-91

JP-A-

1252628

09-10-89

US-A-

5210217

11-05-93

lntcmati. Application No

PCT/EP 94/01714

EP-A-0311768

19-04-89

US-A-

4916075

10-04-90

WO-A-8604926

28-08-86

AU-A-

5516686

10-09-86

CA-A-

1239191

12-07-88

EP-A-

0229765

29-07-87

GB-A-

2173313

08-10-86

JP-T-

62501932

30-07-87

WO-A-8901159

09-02-89

AU-A-

2127988

01-03-89

EP-A-

0382736

22-08-90

JP-T-

3503209

18-07-91

Form PCT.1SA/2I0 (pitcnt fsmtty mnntx) (July 1991)

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